The RB tumor suppressor gene is mutated at high frequency in human cancer, and its encoded protein is an important cell cycle inhibitor and transcriptional regulator. Novel mouse strains with germline mutations in the Rb gene and the related genes p107 and p130 were created using the technique of gene targeting in mouse embryonic stem (ES) cells. The goal of the studies described in this proposal is to establish the role of these genes in normal development and tumorigenesis. Experiments will be conducted using animals or embryos carrying mutations in single genes as well as those with mutations in multiple members of the gene family. Studies on the Rb mutant strain will focus erythropoiesis, specifically the importance of the hepatic microenvironment on the observed erythroid defect in Rb-/-embryos. In addition, the consequences of various combinations of mutations will be examined in regard to the effects on gene expression of other family members, expression of potential target genes and on the profiles of E2F-containing complexes. The phenotype of these mutations will also be characterized in three other specific developmental systems: myogenesis, ocular lens development and lymphopoiesis. The developmental studies of germline mutants will be augmented by the construction of ES cell lines carrying mutations in these genes (again singly or together) and chimeric animals derived from them. Chimeras made in this fashion will also be a source of mutant cells for in vitro analyses. Furthermore, mutant ES cell lines will be used for structure/function analysis of Rb and the different Rb family members through reintroduction of wild type and mutant alleles. Finally, spontaneous tumor development in mice with germline mutations in these genes will be characterized in the presence and absence of p53 function. The information gained from these studies, both in the context of normal development and tumorigenesis, will provide a framework for understanding the function of this important class of genes in human development and disease.